Ejection liquid, ejection method, method of making droplets from liquid, cartridge and ejection device

ABSTRACT

The present invention provides a liquid composition, as an ejection liquid used for stably ejecting liquid droplets, including at least one kind of a protein and a peptide, and a compound having a betaine skeleton by application of thermal energy to the liquid; a method of making droplets form the liquid; and an ejection method and an ejection device suitable for utilizing protein liquid droplets. By adding a compound having a betaine skeleton to an aqueous solution of at least one kind of a protein and a peptide, the liquid composition is improved in stability for ejection by application of thermal energy. Further, a surfactant may be further added to the liquid composition containing the compound having a betaine skeleton, and in this case the effect of stable ejection can be obtained.

This application is a divisional of Application Ser. No. 11/908,600,which was the National Stage of International Application No.PCT/JP2006/307019, filed Mar. 28, 2006. The contents of each of theforegoing applications is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a liquid composition containing atleast one kind of a protein and a peptide suitable for making dropletsform the liquid composition, to a method of making droplets form theliquid composition, and to an ejection device using the method of makingdroplets form the liquid composition.

BACKGROUND ART

In these years, many attempts of using a protein solution as dropletshave been made. Examples of such attempts include a transmucosaladministration as a drug delivery method, and applications to a biochipor a biosensor because they need only a trace amount of protein. Inaddition, a method of using protein liquid microdroplets has alsoattracted attention in the field of screening a bioactive substance(Japanese Patent Application Laid-Open No. 2002-355025; Allain L R, etal., “Fresenius J. Anal. Chem.” 2001, Vol. 371, p. 146-150; and Howard EI, Cachau R E “Biotechniques” 2002, Vol. 33, p. 1302-1306).

Recently, protein, particularly, enzyme or useful protein havingbioactivity is going to be able to be mass-produced through agene-recombination technique so that liquid droplets formation ofprotein can become a useful means for the search and application ofprotein as new medicine, and the applicable field. Above all, the meansof administering various drugs to a patient with the use of the liquidfine droplet has become more important, particularly in the respect ofadministering protein, peptide and other biological substances through alung. Lungs have alveoli with a surface area as large as 50 to 140 m²,have epithelium which is an absorption barrier as extremely thin as 0.1μm, in addition, have enzymatic activity lower than that of thealimentary canal, and accordingly have received attention as asubstituting administration route for the injection of ahigh-molecule-peptide-based drug represented by insulin.

In general, it is known that the intrapulmonary deposition of a liquidfine droplet of a drug largely depends on an aerodynamic particle sizethereof, and above all, in order to deliver the droplet to the alveoli,deep parts of the lung, it is indispensable to develop an administrationform capable of administering droplets having particle sizes of 1 to 5μm and the narrow distribution of the particle sizes with highreproducibility and stable formulation.

There have been conventionally some methods of administering aformulation to the interior of the body particularly to a perimeter of arespiratory organ, so that these methods will be now explained withexamples. There is a metered-dose inhaler (MDI) of aerosolizing theformulation in a suspensoid aerosol form enables quantitativeatomisation, by employing a liquefied incombustible or flame-resistantgas as a pressure carrier, and controlling a unit volume of theliquefied gas to be ejected at a single time. However, the inhaler has aproblem that the size of a droplet in the above-described range is notsufficiently controlled, and besides, the pressure carrier may not begood for health. There is also a spraying method used for atomising aliquid formulation, which employs water and ethanol as a medium, andconverts the liquid formulation to microdroplets by ejecting it togetherwith a gas under pressure for transportation through a capillary.Accordingly, it is theoretically possible for the atomising method tocontrol a atomised amount by specifying a fluid volume of the liquidformulation supplied into such a capillary flow path, but is difficultto control the diameter of the droplet.

Particularly, a spray type of atomisation uses a gas under pressurewhich has been used in a process for making microdroplets from liquid,subsequently as a gas for transporting the atomised microdroplets in aflow. For this reason, it is structurally difficult to vary a quantity(density) of the microdroplets floating in airflow for transportationaccording to a purpose.

As a method of producing the above-described droplets with a narrowparticle size distribution, there is a report on the use of adroplet-generating device which forms extremely microdroplets based on asort of principle used in an ink jet printing (for instance, U.S. Pat.No. 5,894,841 and Japanese Patent Application Laid-Open No.2002-248171). Here, such ink jet system will be described. The systemconsists of the procedure of introducing a liquid to be ejected into asmall chamber, applying pushing force to the liquid, and ejecting thedroplets through an orifice. A usable pushing method includes, forinstance, a method of forming bubbles for ejecting the droplets throughthe orifice on the chamber with the use of an electro-thermal convertersuch as a thin film resistor (thermal ink jet system), and a method ofdirectly pushing a liquid through the orifice on the chamber with theuse of a piezooscillator (piezo ink jet system). The chamber and theorifice are built in a print head element, and the print head element isconnected to a supply source for a liquid and also to a controller forcontrolling the ejection of the droplets.

When making a drug absorbed from lungs, it is necessary to preciselycontrol the dosage particularly for the above-described proteinformulation, so that liquid droplet formation based on the principle ofthe ink jet system is a very preferable form, because it can control anejection rate. In addition, though a liquid is required to be reliablyejected, a protein solution having only adjusted surface tension andviscosity is ejected unstably, so that there was a case where theprotein solution is hardly ejected with a high degree of reproducibilityand efficiency.

The liquid droplet formation of the above-described protein and peptideon the basis of a principle of the ink jet system has a problem that theprotein has a frail spatial configuration and therefore may cause theaggregation and decomposition of the protein when the configuration hasbeen destroyed. When the liquid droplets are formed based on theprinciple of the ink jet system, physical force such as pressure andshear force are applied to the liquid droplets, and each liquid finedroplet has its peculiar high surface energy. They make theconfiguration of most of proteins unstable (When the thermal ink jetsystem is employed, heat is added thereto in addition to them). Theliquid droplet formation particularly on the basis of the principle ofthe ink jet system has the problem that storage in a long period isunstable, and further that the above-described physical force isextremely higher than the shear force and thermal energy applied innormal stirring and heat treatment. (It is considered that, forinstance, the instantaneous load of 90 atmospheres at 300° C. is appliedto the liquid droplets in the thermal ink jet system). In addition, aplurality of physical forces are simultaneously applied to the liquiddroplets. For this reason, protein tends to become much more unstablethan in a process of normally treating protein, so that there has been acase where a conventionally-used technology of stabilizing the proteinis insufficient. Once the problem happens, proteins aggregate while thedroplets are formed, which causes clogging in a nozzle and makes thedroplets hardly ejected.

Furthermore, a size of a droplet suitable for lung inhalation is 1 to 5μm, which is very smaller than a droplet of about 16 μm used in acurrently commercially available printer, and results in applying largersurface energy and shear force onto the droplet. For this reason, it isextremely difficult to eject protein as microdroplets suitable for thelung inhalation.

In consideration of the above-described various uses, a method ofejecting a protein solution is preferably based on the principle of athermal ink jet system, because it has a low manufacturing cost and canincrease the density of nozzles.

On the other hand, a method of adding a water-soluble polymer oralbumin, such as a surfactant, glycerol, various saccharides andpolyethylene glycol, which are known as the method of stabilizingprotein, has little or no effect of improving ejecting performance whenejecting protein with a thermal ink jet system.

In regard to a liquid composition of droplets to be inhaled into lungs,which are formed with the use of a thermal ink jet system, there isdisclosed a method of adding a compound for adjusting surface tensionand/or a moisturizing agent to a protein solution (for instance, thepamphlet of International Publication No. WO02/094342). The above methodincludes adding a water-soluble polymer such as a surfactant andpolyethylene glycol to a protein solution, because describing that thewater-soluble polymer improves the stability of protein in a solution ofa formed liquid droplet, through decreasing surface tension andviscosity of the solution, and keeping the moisture of the solution.

However, the pamphlet does not describe the stability of ejection,furthermore, the addition of a surfactant and a water-soluble polymershows the insufficient effect when the concentration of protein andpeptide is high, and there was a case where an additive in itselfaggravated the stability of the ejection. In addition, many surfactantsdo not have an effect on the stability of the ejection at all, and inother words, surface tension, viscosity or a moisture retention effectdoes not control the stability of the ejection. To put it differently,the above-described method was not a general one for stabilizing theejection when ejecting protein and peptide with a thermal ink jetsystem.

As described above, an ink jet technology is well known as a method ofmaking liquid fine droplets from a liquid sample and ejecting them, andparticularly has a feature of showing high controllability for even atrace amount of a liquid to be ejected after having converted intoliquid droplets. The fine-droplet-ejecting type of an ink jet systemincludes an oscillation type using a piezoelectric element and a thermalink jet system using a micro-heater element. The oscillation type usingthe piezoelectric element has limitation in miniaturization for thepiezoelectric element to be used, and accordingly in the number ofinstalled ejection orifices per unit area. In addition, a necessary costfor producing an ejection device sharply increases with the increase ofthe number of arranged ejection orifices per unit area. In contrast tothis, the thermal ink jet system can comparatively easily miniaturizethe micro-heater elements to be used in the ejection device, canincrease the number of the arranged ejection orifices per unit area incomparison with the oscillation type system using the piezoelectricelement, and can far reduce a necessary production cost for the ejectiondevice.

When applying a thermal ink jet system to liquid droplet formation, itis necessary to adjust a physical property of a liquid to be ejected, soas to control an appropriate atomisation state and a liquid volume of aliquid fine droplet to be ejected from each ejection orifice.Specifically, a liquid composition such as a type of a solvent, acomposition and a concentration of a solute, which composes a liquidsample to be ejected is well adjusted so as to provide a target liquidvolume of a liquid fine droplet.

Furthermore, various technological developments are being proceeded alsoon a droplet-ejecting mechanism based on the principle of a thermal inkjet system. While a conventional ink jet printer head ejects liquiddroplets having the individual liquid volume of about severalpicoliters, an ejecting technology and an ejecting mechanism which havebeen developed recently forms an extremely liquid fine droplet of asubpicoliter or femtoliter order (see, for instance, Japanese PatentApplication Laid-Open No. 2003-154655).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a liquid composition asan ejection liquid used for stably ejecting liquid droplets containingat least one kind of a protein and a peptide by application of thermalenergy to the liquid, and to provide an ejection method and an ejectiondevice suitable for utilization of protein liquid droplets.

The ejection liquid of the present invention is characterized byincluding one kind selected from a protein and a peptide, a compoundhaving a betaine skeleton, and a liquid medium.

The ejection method of the present invention is characterized in thatliquid droplets are made from the above-described ejection liquid basedon the principle of the thermal ink jet system, and the liquid dropletsare ejected.

The cartridge for ejecting a liquid according to the present inventionis characterized by comprising a tank for accommodating theabove-described ejection liquid, and an ejection head based on thethermal ink jet principle.

The liquid inhalation device of the present invention is characterizedby comprising the above-described cartridge, and a flow path part andopening part for guiding a liquid to be ejected, from a liquid ejectionpart of an ejection head of the cartridge based on the thermal ink jetprinciple, to an inhaling site of a user.

The method of making liquid droplets from a liquid according to thepresent invention is characterized in that the method makes liquiddroplets from a liquid containing at least one kind of a protein and apeptide by applying thermal energy to the liquid, wherein theabove-described liquid includes a compound having a betaine skeleton.

According to the present invention, an ejection liquid which can bestably ejected after thermal energy has been applied thereto can beprovided, by adding a compound having a betaine skeleton to a solutioncontaining at least one kind of a protein and a peptide. In addition, itis further possible to eject a protein solution with a higherconcentration by further adding a surfactant to the ejection liquid,because the addition provides a synergistic effect of stabilizingejection. When at least one kind of a protein and a peptide is apharmaceutically effective ingredient, at least one kind of the proteinand peptide of the pharmaceutically effective ingredient arrives at alung by ejecting the ejection liquid from a portable ejection device toconvert it into liquid droplets, and inhaling the droplet, and thepharmaceutically effective ingredient can be absorbed by the lung. Theabove-described method can be also used for the preparation of a biochipor a biosensor, sensing, and screening for a biological substance, byejecting at least one kind of the protein and peptide onto a substratewith the method.

Other features and advantages of the present invention will be apparentfrom the following description taken in conjunction with theaccompanying drawings, in which like reference characters designate thesame or similar parts throughout the figures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view for explaining a method for ejectingprotein onto a substrate;

FIG. 2 is a view for showing one example of a pattern of proteinarranged on a substrate;

FIG. 3 is a diagrammatic explanatory drawing of a head cartridge unitfor an inhaler;

FIG. 4 is a perspective view of an inhaler;

FIG. 5 is a perspective view for a state in which an access cover isopened in FIG. 4;

FIG. 6 is a graph showing an ejection rate when an albumin solution isejected with the thermal ink jet system; and

FIG. 7 is a model view of an experimental procedure in Example 20.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention will now be described indetail in accordance with the accompanying drawings.

An object of the present invention is to provide a liquid compositionfor an ejection liquid used for stably ejecting liquid dropletscontaining at least one kind of a protein and a peptide by applyingthermal energy to it, to provide an ejection method suitable for the useof the liquid droplet containing protein, and to provide an ejectiondevice therefor.

The present invention will be now described in detail below.

The protein used in the present invention means an arbitrary polypeptidewhich consists of amino acids bonded with each other by a peptide bond,and can be dissolved or dispersed in an aqueous solution. In addition,the peptide used in the present invention means a compound whichconsists of two or more and 100 or less amino acids bonded with eachother through a peptide bond. Protein and peptide may be chemicallysynthesized or refined from a natural source, but typically, are nativeprotein and a recombinant of peptide. Protein and peptide can be alsochemically reformed by covalently bonding polyethylene glycol with anamino acid residue in the molecule of protein and peptide or the like,to enhance an effect of prolonging a therapeutic effect of protein andpeptide and the like.

When implementing the present invention, a liquid to be used can containvarious protein and peptide of which the liquid droplet is desired to beformed. Most typically, the purpose of making the liquid droplet fromthe liquid containing protein and peptide of the present invention is todeliver useful protein and peptide for medical treatment to a lung.These examples include various hematopoietic factors such as calcitonin,a blood coagulation factor, ciclosporin, G-CSF, GM-CSF, SCF, EPO, GM-MSFand CSF-1; interleukin such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11 and IL-12; an IGF; and cytokine includingM-CSF, thymosin, TNF and LIF. Usable protein having another therapeuticeffect includes vasoactivity peptide; interferon (alpha, beta, gamma orcommon interferon); a growth factor or a hormone, such as a human growthhormone and another animal growth hormone (like growth factors ofbovine, pig or chicken); insulin; oxytocin; angiotensin; methionineenkephalin; substance P; ET-1; FGF; KGF; EGF; IGF; PDGF; LHRH; GHRH;FSH; DDAVP; PTH; vasopressin; glucagon; and somatostatin. A proteaseinhibitor such as leupeptin and pepstatin, and a metalloproteinaseinhibitor (such as TIMP-1, TIMP-2 and other proteinase inhibitors) arealso used. A nerve growth factor such as BDNF and NT3 is also used. Aplasminogen activator such as tPA, urokinase and streptokinase is alsoused. A peptide part of protein having a main structure or one part ofparent protein and at least one part of various biological properties ofparent protein is also used. An analog such as a substitution or defectanalog, modified amino acid such as a peptide analog, or a compoundincluding the above-described substance modified with a water-solublepolymer such as PEG and PVA is also used. It has been clear in CriticalReviews in Therapeutic Drug Carrier Systems, 12 (2&3) (1995) that theabove-described protein can be delivered to a lung.

Furthermore, in a field of the preparation of a biochip and a biosensor,the screening of protein and peptide and the like, the following manysubstances in addition to the above-described protein and peptide can bealso used: various enzymes such as oxidase, reductase, transferase,hydorase, lyase, isomerase, synthetase, epimerase, mutase and racemase;various antibodies and receptors such as IgG and IgE; protein andpeptide used for diagnosis, such as antigen of them, allergen,chaperonin, avidin and biotin; and the above-described substancesmodified with a reagent for immobilization.

Usable protein and peptide contained in the above-described ejectionliquid has a molecular weight, for instance, in a range of 0.5 kDa to150 kDa. In addition, a content of at least one kind selected fromprotein and peptide is determined in accordance with a purpose andapplication thereof, and is preferably determined in a range of 1 ng/mlto 200 mg/ml.

It is preferable that a liquid medium contains mainly water, and that aratio of water to the medium is 50% or higher. In addition to waterwhich is a main component of the medium, a water-soluble organic solventand an auxiliary such as alcohol can be added as the medium.

It is generally known to add a surfactant and a solvent such as ethyleneglycol, in order to improve ejection properties of ink to which thermalenergy is applied. However, when ejecting a protein and peptidesolution, the ejection properties were not perceptibly improved byadding only them, so that a new additive was necessary.

As a result of conducting extensive studies, the present inventors foundthat a solution containing a compound having a betaine skeleton in aprotein and peptide is suitable for forming a stable liquid droplet evenwhen thermal energy has been applied.

As a method for applying the above-described thermal energy to liquiddroplets and ejecting them, there are, for instance, a method of heatinga tube and ejecting a liquid inside it from an opening, which isdisclosed in U.S. Pat. No. 6,234,167, and a method based on theprinciple of the thermal ink jet method. The thermal ink jet system isthe method of applying the thermal energy to the liquid with a heater,heating and bubbling the liquid with the energy, and ejecting the liquiddroplet from the opening of an end of a liquid-existing space; and has afeature of uniformly ejecting the highly accurate number of liquiddroplets in comparison with the above-described method of heating thetube, by separating the heaters into fine many parts.

Hereafter, a configuration based on the principle of the thermal ink jetmethod is mainly described because the thermal ink jet method mostconspicuously shows an improved effect for ejection properties, but thepiezoelectric ink jet method of ejecting a liquid in a nozzle by using avibratory pressure due to a piezoelectric element can be used in thepresent invention. The thermal ink jet system can enhance: the accuracyof a diameter of an ejection orifice and the heat quantity of a heatpulse used for ejection in an individual ejection unit for liquidformulation, and the accuracy of a size of a micro heater or the likeused therefor; and the reproducibility. Accordingly, the thermal ink jetmethod can achieve a narrow distribution of the diameters of liquiddroplets over the all of many ejection units for a liquid formulation,which are densely arranged on an ejection head. In addition, in asituation in which the present invention is frequently used, the deviceis required to satisfy demands that a manufacture cost is low, the headmust be changed frequently, and the device is small, and then thethermal ink jet system is further preferably used.

The present inventors consider the reason why a compound having abetaine skeleton so greatly contributes to the ejection stability, inthe following way. A betaine skeleton has both of a quaternary ammoniumcation and an organic acid anion in a near position to each other in onemolecule, and has features of: tending to be very easily hydrated; beingeasily modified by other molecules and being able to have an alkyl groupor acyl group of a long chain in the molecule; and therefore tending toshow high hydration property even when a compound having the betaineskeleton also has the alkyl group of the long chain. On the other hand,protein and peptide are strongly hydrophobic and are difficult to becomestable by hydration. When the compound having a betaine skeleton has ahydrophobic group such as the above-described alkyl group and acyl groupwith a long chain, these functional group acts on a hydrophobic site inprotein or peptide, and at the same time the cation and anion of thebetaine skelton hydrate the protein and the peptide by the hydrationforce of the cation and anion having high hydration property, tostabilize them and inhibit interaction between proteins with each otherand between peptides with each other. By the action, the compound havinga betaine skeleton can inhibit the protein and the peptide from causingdenaturation and aggregation due to an energy load when the liquid isejected based on the principle of the thermal ink jet method, and canstabilize the ejection.

A compound having a betaine skeleton to be used in the present inventionhas preferably a chemical structure as is represented by the followingformula (1).

Here, R₁ in the formula (1) is a substituted or unsubstituted alkylgroup having 6 to 18 carbon atoms, and more preferably a saturated alkylhaving 8 to 16 carbon atoms. In the formula (1), R₂ and R₅ are eachindependently a substituted or unsubstituted alkylene chain having 1 to6 carbon atoms, and more preferably and particularly, an alkylene chainhaving 1 to 4 carbon atoms. In the formula (1), R₃ and R₄ are eachindependently an alkyl group or an alkylene chain each having 1 to 6carbon atoms, and R₃ and R₄ may be bonded together to form aheterocyclic ring.

An example of the above-described compound includesdimethyldialkylbetaine, diethyldialkylbetaine andmethylethyldialkylbetaine; and imidazolium betaine having a heterocyclicring as well represented by the following formula (2).

In the formula 1 and the formula 2, A is an anion of an organic acid,and is more preferably a carboxylic group or a sulfonic group. When A isa sulfonic group, R₅ has preferably a hydroxyl group.

In the formula (1) and the formula (2), X₁ and X₂ are counter ions, andX₁ has only to be an anionic species, and has only to have at least oneselected from inorganic and/or organic anions. An example of the counterion of X₁ preferably includes a halide ion, a chloride ion, a bromideion, an iodide ion, a fluoride ion, a hydroxide ion, a carboxylic acidion, a nitric acid ion, a phosphoric acid ion and a sulfuric acid ion;and the counter ions may be the same or different from X₂. X₂ has onlyto be a cationic species, and represents at least one selected from amonovalent metal ion, a metal oxide ion and an organic cation. Thecounter ion of X₂ may be the same as or different from X₁.

In the formula (1), n is the repetition number of the skeleton, and is 0or 1. When n is 0, the compound is alkylbetaine shown in the formula(3); and when n is 1, the compound is alkylamide alkylbetaine shown inthe formula (4) or the formula (5).

A compound having a betaine skeleton used in the present invention caninclude alkylamide alkylbetaine, a salt thereof, and a derivativethereof in such a range that the effect of the present invention is notdeteriorated, and preferably alkylamide alkylbetaine is used.

The ejection liquid of the present invention is prepared, though beingnot particularly limited to the following procedure, by mixing acompound having betaine skeleton having surface-active properties and atleast one kind of a protein and a peptide with a liquid having acomposition composed of a liquid medium mainly containing water andother additive components, which are the above-described components ofthe ejection liquid. The form of a liquid mixture is not particularlylimited, and may be any of a solution type, a suspension type, anemulsion type and a dispersion type. When the liquid mixture is not thesolution type, a usable size of a suspended matter, an emulsified matteror a dispersed matter in a medium is in a range of a subnanometer scaleto a micrometer scale.

In the present invention, the present inventors have further found thatit is possible to keep the stability of ejection even when theconcentration of an additive is largely decreased, by adding asurfactant together with a compound having a betaine skeleton. Theaddition of 0.2 to 1 part by weight of the surfactant with respect to 1part by weight of a compound having the betaine skeleton can reduce anamount of a compound having the betaine skeleton to be added, withrespect to a solution having the same concentration of protein, into1/10 to 1/2 while keeping the stability of the ejection.

The effect of a surfactant is considered to be different from effect ofa compound having a betaine skeleton, and to stabilize ejection throughthe action of inhibiting the denaturation of protein and the action ofredissolving protein which has once aggregated. It is considered thatthe combination of these two different effects develops a synergisticeffect to greatly improve the stabilization of ejection. It isconsidered that the single addition of a surfactant could not completelyinhibit the aggregation of protein because the surfactant alone has nolarge effect, whereby it could not secure the stability of ejection.

A surfactant of the present invention means a compound which has both ofa polar part and a nonpolar part in one molecule, has each of theabove-described parts in a distant region from each other in themolecule, and has a property capable of reducing an interfacial tensionbetween two immiscible phases by alignment of the molecular of thesurfactant between the two immiscible phases and capable of forming amicell.

Examples of the surfactant that can be used typically includes, but isnot limited to, sorbitan fatty acid esters such as sorbitanmonocaprylate, sorbitan monolaurate and sorbitan monopalmitate; glycerinfatty acid esters such as glycerin monocaprylate, glycerin monomyristateand glycerin monostearate; polyglyceryl fatty acid esters such asdecaglyceryl monostearate, decaglyceryl distearate and decaglycerylmonolinoleate; polyoxyethylene sorbitan fatty acid esters such aspolyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate andpolyoxyethylene sorbitan tristearate; polyoxyethylene sorbitan fattyacid esters such as polyoxyethylene sorbit tetrastearate andpolyoxyethylene sorbit tetraoleate; polyoxyethylene glycerin fatty acidesters such as polyoxyethylene glyceryl monostearate; polyethyleneglycol fatty acid esters such as polyethylene glycol distearate;polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ethers;polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether and polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylenealkyl phenyl ethers such as polyoxyethylene nonylphenyl ether,polyoxyethylene hydrogenated castor oils such as polyoxyethylene castoroil and polyoxyethylene hydrogenated castor oil (polyoxyethylenehydrogen castor oil); polyoxyethylene beeswax derivatives such aspolyoxyethylene sorbit beeswax; polyoxyethylene lanolin derivatives suchas polyoxyethylene lanolin; polyoxyethylene stearic acid amides havingHLB of 6 to 18 out of polyoxyethylene fatty acid amides; anionicsurfactants including alkyl sulfate containing an alkyl group having 8to 18 carbon atoms such as sodium cetyl sulfate, sodium lauryl sulfateand sodium oleyl sulfate, and polyoxyethylene alkyl ether sulfatescontaining 2 to 4 moles by average of ethyleneoxide added and an alkylgroup having 8 to 18 carbon atoms such as polyoxyethylene sodium laurylsulfate; an alkylbenzene sulfonates containing an alkyl group having 8to 18 carbon atoms such as sodium lauryl benzenesulfonate; alkylsulfosuccinates containing an alkyl group having 8 to 18 carbon atomssuch as sodium lauryl suifosuccinate; natural surfactants such aslecithin and glycerophospholipid; sphingophospholipids such assphingomyelin; and saccharose fatty acid esters of a fatty acid having 8to 18 carbon atoms. An ejection liquid (liquid composition) of thepresent invention can contain one or more of these surfactants incombination.

The surfactant is preferably polyoxyethylene sorbitan fatty acid ester,is particularly preferably polyoxyethylene 20 sorbitan monolaurate,polyoxyethylene (4) sorbitan monoolate, polyoxyethylene 20 sorbitanmonopalmitate, polyoxyethylene 20 sorbitan monostearate, polyoxyethylene20 sorbitan tristearate, polyoxyethylene 20 sorbitan monolaurate,polyoxyethylene (5) sorbitan monooleate and polyoxyethylene 20 sorbitantri-oleate, and is most preferably polyoxyethylene 20 sorbitanmonolaurate and polyoxyethylene 20 sorbitan monooleate. In addition, thesurfactant particularly preferable for lung inhalation ispolyoxyethylene 20 sorbitan monolaurate and polyoxyethylene 20 sorbitanmonooleate.

A concentration of a surfactant to be added can be, for instance, 0.001to 20% by weight in the case of insulin, though depending on coexistingprotein or the like. The content of a surfactant to be added ispreferably 0.2 to 10 parts by weight, with respect to 1 part by weightof a compound having a betaine skeleton.

In an embodiment of the present invention, an antibacterial agent, adisinfectant and an antiseptic agent may be added in order to eliminatethe influence of a microorganism. A compound having a betaine skeletonto be used in the present invention has the above-described effect, butthe agent having the effect further includes, for instance, a quaternaryammonium salt such as benzalkonium chloride and benzathonium chloride; aphenol derivative such as phenol, cresol and anisole; benzoic acids suchas benzoic acid and p-hydroxybenzoate ester; and sorbic acid.

In an embodiment of the present invention, an ejection liquid mayinclude oil, glycerin, ethanol, urea, cellulose, polyethylene glycol andalginate, so as to increase the physical stability for cohesion andprecipitation occurring while being preserved; and may include ascorbicacid, citric acid, cyclodextrin, tocopherol and other antioxidantalagents, so as to increase the chemical stability for preventingdeterioration and oxidation.

An ejection liquid may include a buffer agent for adjusting pH. A usablebuffer solution includes, for instance, ascorbic acid, citric acid,diluted hydrochloric acid, diluted sodium hydroxide, further sodiumhydrogenphosphate, sodium dihydrogenphosphate, potassiumhydrogenphosphate, potassium dihydrogenphosphate, PBS, Hepes and Tris.

An ejection liquid may include an isotonizing agent such as aminoethylsulfonic acid, potassium chloride, sodium chloride, glycerin and sodiumhydrogen carbonate.

An ejection liquid may include a corrigent for taste and smell such assaccharide like glucose and sorbitol, a sweetening agent like astelpalm, menthol and various flavors. In addition, the usable corrigent maybe not only a hydrophilic compound, but also a hydrophobic compound oran oily compound.

When the ejection liquid of the present invention is used for preparinga biochip and a biosensor or screening protein, a system which isapproximately similar to a currently commercially available ink jetprinter can be used.

A method of ejecting protein with the use of the ejection liquid of thepresent invention will be now described in detail with reference toFIG. 1. A pattern is formed by the steps of: filling the ejection liquidinto a nozzle of an ink jet head 3 from a tank 1; and making the ink jethead eject the ejection liquid onto a substrate 5 suitable for eachpurpose, by driving the ink jet head while keeping a fixed space betweenthe substrate and the nozzle face of the ink jet head. In FIG. 1,reference numeral 2 denotes a liquid flow path, reference numeral 4denotes liquid droplets, and reference numeral 6 denotes a drivecontroller. It is recommended to use a drive controller when ejectingthe ejection liquid so as to form the pattern according to an imagepattern on the substrate, and it is preferable to form such a patternthat spots are not connected with each other as shown in FIG. 2.

An ejection liquid in the present invention can include variousadditives adaptable to a purpose of an application of an atomisationliquid, for instance, a proper amount of a surface moderator, aviscosity modifier, a solvent and a moisturizing agent, as needed.

Specifically, a blendable additive includes, for instance, a hydrophilicbinder, a hydrophobic binder, a hydrophilic thickener, a hydrophobicthickener, glycol derivatives, alcohols, a corrigent for taste, acorrigent for smell and an electrolyte, which may be a single substanceor a mixture selected from them.

Here, various substances to be used for the above exemplified additivesare more preferably permitted to use in a medicinal use described in apharmacopoeia of each country as a secondary component which may beadded when preparing a liquid formulation for therapy, or in a foodproduct and a cosmetic article.

Various substances to be blended as the above-described additive arepreferably added generally each in a range of 0.01 to 40% by a weightratio, and more preferably in a range of 0.1 to 20%, though the valuedepends on a type of objective protein and peptide. In addition, anamount of the above-described additive to be added is preferably 0.5 to200 parts by weight with respect to 1 part by weight of theabove-described protein and peptide from a viewpoint of an ejectionproperty, though the amount varies with a type, amount and combinationof the additives.

An atomiser of the present invention has preferably a structure havingan ejection head part which can eject a liquid fine droplet of theliquid formulation while applying thermal energy to the liquidformulation and works based on a thermal ink jet principle, and manyejection units for the liquid formulation, which compose the head partand can be independently driven. Furthermore, the atomiser preferablyhas a form provided with: an electrical connection part used forconnecting a plurality of control signals required for independentlydriving each of the ejection units for the liquid formulation,integrated with a wire for connecting the ejection units for the liquidformulation with each other; in addition, a tank for accommodating theabove-described liquid formulation; and an integrally composed cartridgefor nebulizing a liquid while including a liquid flow path, as means forsupplying the liquid formulation from the tank to the ejection headbased on the thermal ink jet principle.

FIG. 3 diagrammatically shows an example of the whole configuration ofsuch a cartridge for nebulizing a liquid. The cartridge illustrated inFIG. 3 is produced by arranging a head part 9 for nebulizing a liquidformulation, a tank 7 for filling the liquid formulation therein, and aliquid flow path 8 for introducing the liquid formulation from the tank7 to the head part 9, integrally on the same substrate. A controller forcontrolling the driving of each ejection unit for a liquid formulationin the head part 9 exchanges a driving signal, a control signal and thelike with the head part 9, through an electrical connection part 11 towhich an inner wire 10 is connected.

In the above arrangement, a head part 9 preferably employs the head forejecting a liquid ultrafine droplet, which ejects liquid droplets in anindividual liquid amount of a subpicoliter or femtoliter order, hassuperior controllability for the liquid amount and is disclosed inJapanese Patent Application Laid-Open No. 2003-154655.

In examples shown in FIG. 1 and FIG. 3, one type of a liquid formulationis atomised, so that there is one tank for filling the liquidformulation therein in the structure. When two or more types of liquidformulations are atomised, it is possible to cope with the case byarranging a plurality of tanks for filling the liquid formulationsappropriately corresponding to them, and making a thermal ink jet headhave a configuration of having integrated a plurality of ejection unitsfor the liquid formulations.

When ejecting the ejection liquid of the present invention onto asubstrate with the use of the ink jet system, it is also possible toefficiently react the substrate with a substance to be detected, byejecting a solution containing the substance to be detected onto thesubstrate in the same pattern, and to change the concentration ofprotein by only changing an amount to be ejected. Accordingly, theatomiser can be used for preparing a biochip, a biosensor, and a devicefor sensing and screening a biological substance. Furthermore, theatomiser is effectively used through employing a head capable ofejecting the above-described liquid fine droplet, when it is required touse the above-described extremely microdroplet as an individual liquiddroplet to be ejected, for instance, when a somatic cell with a size ofseveral micrometers has to be applied as a drug.

An inhalation device of the present invention makes use of an advantagepeculiar to the form in the inhalation device, which separates a processof transforming a liquid formulation to a liquid fine droplet from aprocess of mixing the atomised liquid fine droplet into an airflow fortransporting the liquid fine droplet, which is a feature of a nebulizingmethod of the present invention. When nebulizing a liquid formulationcontaining an ejection liquid (liquid composition) in a predeterminedconcentration, which is specified in the present invention and can beused for a therapeutic purpose, into an airflow, and when making aperson to be administered inhale the atomised liquid formulation, theinhalation device can arbitrarily set an amount of a drug compound (doseper single dosage) in the liquid composition contained in a gas to beinhaled, which can be used for the therapeutic purpose. The inhalationdevice to be used for the above purpose can be miniaturized so that auser can carry and hold it, by using an ejection head based on thethermal ink jet principle, which arranges openings for ejecting liquidfine droplets therein at high density per unit area, as a nebulizingmechanism for nebulizing the above-described liquid formulation.

In the inhalation device which uses the above-described ejection liquidfor lung inhalation, an essential part is a device which can eject aformulated substance of the present invention in a form of liquiddroplets with particle sizes of 1 to 5 μm and with a narrowdistribution. A head part for ejecting the liquid droplet is a removablecartridge unit which was described in the above with reference to FIG.3.

An inhalation device as a controller for ejection is composed so that auser can carry and hold, and an inhaler can make a user inhale a drug ina form of liquid droplets which are ejected so as to acquire a uniformparticle size and a constant amount.

An outline of an example for an inhaler usable in the present inventionwill be now described with reference to FIG. 4 and FIG. 5.

FIG. 4 is a perspective view showing an appearance of an inhaler,reference numeral 15 denotes a main body of the inhaler, and referencenumeral 12 denotes an access cover, both of which form a housing.Reference numeral 14 denotes a power button. FIG. 5 illustrates a statein which the access cover 12 is opened. When the access cover 12 isopened, a head cartridge unit 16 and a mouthpiece 13 appear. When a userstarts an inhalation operation, air flows into a mouthpiece 13 from anair intake, is mixed with a drug ejected from an ejection openingprovided in a head part 9 of the head cartridge unit 16 to form a fluidmixture, and flows to an outlet of the mouthpiece which has a shape tobe taken in a mouth of a human. The user can effectively inspire a drugsolution ejected as liquid droplets from a liquid-ejecting part of ahead cartridge unit, through inserting a tip of the mouthpiece insidethe mouth, holding it with a tooth, and inhaling air.

In other words, a configuration of the intake part corresponds to aninhalation mechanism for making a person to be administered inhale a gasin which liquid fine droplets of a liquid formulation produced by anebulizing mechanism float in a mist form.

FIG. 4 and FIG. 5 show a configuration of an example of an inhaler to beused for a medical purpose, which is miniaturized so that a user cancarry and hold it. A main body of the inhaler 15 consists of a housingfor accommodating a cartridge for nebulizing liquid, a controllerthereof and a power source (battery); and a mouthpiece 13 to cover amouth when inhaling air, mounted thereon. The cartridge for nebulizingliquid is integrated with a tank for a liquid formulation as isillustrated in FIG. 3, and has such a configuration that it can beexchanged after an access cover 12 has been opened. FIG. 5 illustrates astate in which the access cover 12 is opened. A head cartridge unit 16is installed on some midpoint of a tubular airflow path for introducingair which flows in from an air intake opening, to a mouthpiece 8therethrough. A head part of the head cartridge unit 16 converts aliquid formulation into liquid fine droplets based on the principle ofthe thermal ink jet system, and the tubular airflow path mixes them inairflow therein, and atomises them. The inhaler employs a system ofintroducing air inside it from the air intake opening, when a user takesthe mouthpiece 13 in a mouth and inhales air through the mouthpiece.

By adopting a configuration shown in FIG. 5, the inhaler achieves a formwhich can naturally make atomised liquid fine droplets of a liquidformulation arrive at a fauces and inside the trachea of a person to beadministered, together with inspired air. Accordingly, an amount (dose)of the liquid formulation to be atomised does not depend on varyingvolumes of the inspired air, and is independently controlled.Specifically, a head part of a head cartridge unit 16 employs a head forejecting the liquid ultrafine droplet disclosed in Japanese PatentApplication Laid-Open No. 2003-154655, and has a configuration ofcontrolling the diameter of the droplets to about 3 μm by average.

EXAMPLES

First of all, in order to further promote understanding for difficultyin the ejection of a protein solution, an ejected amount of protein whenonly protein has been ejected with the thermal ink jet system will beshown. A protein solution was prepared by dissolving albumin in PBS, andthe solution of each concentration was ejected by using a thermal inkjet printer (trade name: PIXUS950i, manufactured by Canon Inc.) whichhas been remodeled so that the ejected solution can be recovered. Theamount of ejected each albumin solution was expressed by values withrespect to 100% of the ejected amount of pure water which has beenejected in the same manner. The results are shown in FIG. 6.

It is understood that a solution containing albumin even as in a lowconcentration as 1 μg/mL is not completely stably ejected, and as asolution contains more protein, it is slowly hardly ejected. Whencarrying out such as operation as is required in the present invention,liquid droplets with a smaller diameter has to be ejected, and it isconceivable that a protein solution is hardly ejected.

The present invention will be now described in further detail withreference to the following Examples, but Examples are specific examplesshown for deeper understanding, and the present invention is not limitedto these specific examples at all. Hereafter, “%” means % by weight.

Examples 1 to 12 and Comparative Examples 1 to 13

(Confirmation of Diameter of Atomised Particle)

An aqueous solution containing 30% of ethanol was filled in a headcartridge having a nozzle diameter of 3 μm to be used in an ejectionexperiment, and a size and size distribution of the ejected particleswere confirmed by measuring them with the use of alaser-diffraction-type particle size distribution measuring instrument(Spraytec manufactured by Malvern Instruments Ltd.). As a result, it wasdetected that the ejected particles are certainly liquid droplets havinga sharp particle size distribution at 3 μm.

(Liquid Droplet Formation from Protein Solution Based on Principle ofThermal Ink Jet System)

An ejection liquid was prepared by the steps of: previously dissolvingan appropriate concentration of albumin in purified water; furtheradding a compound having a betaine skeleton while stirring the solution;and then quantitatively determining the volume with purified water sothat the concentration of each substance can have its desiredconcentration.

A prepared ejection liquid was ejected by the steps of: filling theejection liquid in the above-described head cartridge having the nozzlediameter of 3 μm; connecting the head cartridge with an ejectioncontroller; setting the frequency to 20 kHz and the voltage to 12 V;ejecting the ejection liquid for one second; and again ejecting it afterthe interval of 3 seconds. The ejection liquid was ejected based on theprinciple of the thermal ink jet system. The ejection operation wasrepeated for 50 times and it was visually confirmed whether the ejectionliquid was ejected, or not. The ejection liquid which was ejected for 50operations was evaluated as “∘”, the ejection liquid of 15 to 49operations as “Δ”, and the ejection liquid of less than 15 operations as“×”. In addition, the atomised ejection liquid was recovered, and it wasconfirmed whether the composition changed or not, by analyzing theejection liquids before and after ejection, through an HPLC analysis (ina measurement condition of apparatus: JASCO Corporation; column:YMC-Pack Diol-200, 500×8.0 mm ID; eluent: a liquid including 0.1MKH₂PO₄—K₂HPO₄(pH 7.0) 0.2M NaCl; flow rate: 0.7 mL/minute; temperature:25° C.; detection: UV 215 nm). An ejection test shown below was carriedout in the above-described conditions, with the use of a head cartridgehaving the above-described nozzle with the diameter of 3 μm and anejection controller.

As a comparative example, an ejection liquid was prepared which containspure water, various protein solutions and substances having nothing todo with the present invention, and was subjected to an ejectionexperiment as in the case of the present examples. Formulations andresults examined on Examples and Comparative Examples were listed in thefollowing Table 1.

TABLE 1 Ejection Protein Compound having betaine skeleton Surfactant andadditive property Type Concentration Type Concentration TypeConcentration Evaluation Example 1 albumin 1 mg/ml lauramidepropylbetaine 10 mg/ml none — ∘ Example 2 albumin 1 mg/ml cocamidepropylbetaine 10 mg/ml none — ∘ Example 3 insulin 4 mg/ml cocamidepropylbetaine 10 mg/ml none — ∘ Example 4 insulin 4 mg/ml lauramidepropylbetaine 10 mg/ml none — ∘ Example 5 insulin 4 mg/ml lauryl betaine 50mg/ml none — ∘ Example 6 glucagon 0.5 mg/ml   cocamidepropyl betaine  3mg/ml none — ∘ Example 7 GLP-1 1 mg/ml cocamidepropyl betaine 10 mg/mlnone — ∘ Example 8 hGH 1 mg/ml cocamidepropyl betaine 10 mg/ml none — ∘Example 9 EPO 1 mg/ml lauramidepropyl betaine 10 mg/ml none — ∘ Example10 IFN α 1 mg/ml lauramidepropyl betaine 10 mg/ml none — ∘ Example 11IFN γ 1 mg/ml lauramidepropyl betaine 10 mg/ml none — ∘ Example 12calcitonin lauramidepropyl betaine 10 mg/ml none — ∘ Comparative Example1 water — none — none — ∘ Comparative Example 2 albumin 1 mg/ml none —none — x Comparative Example 3 insulin 4 mg/ml none — none — xComparative Example 4 glucagon 0.5 mg/ml   none — none — x ComparativeExample 5 GLP-1 1 mg/ml none — none — x Comparative Example 6 hGH 1mg/ml none — none — x Comparative Example 7 EPO 1 mg/ml none — none — xComparative Example 8 IFN α 1 mg/ml none — none — x Comparative Example9 IFN γ 1 mg/ml none — none — x Comparative Example 10 calcitonin 1mg/ml none — none — x Comparative Example 11 albumin 1 mg/ml none —TWEEN80 10 mg/ml x Comparative Example 12 insulin 4 mg/ml none — TWEEN2010 mg/ml Δ Comparative Example 13 insulin 4 mg/ml none — TWEEN20 50mg/ml x

Pure water of Comparative Example 1 was stably ejected because ofcontaining no protein, whereas ejection liquids of Comparative Examples2 to 13, which does not contain a compound having a betaine skeleton,was ejected little or was not ejected at all, regardless of the type ofthe protein and the presence or absence of an additive. Liquids ofComparative Examples 11 to 13 which contain a surfactant TWEEN wereejected to some extent, but was not sufficiently stably ejected. It isunderstood that in contrast to this, Examples 1 to 12 are normally andstably ejected. As a result of an HPLC analysis, Examples 1 to 12 didnot show a change of a peak location and a peak area before and afterejection, and a change of a liquid composition, either.

Examples 13 and 14

(Effect onto Each Protein, and Concentration of Additive)

Subsequently, lauramidepropyl betaine or cocamidepropyl betaine wasselected as a compound having a betaine skeleton and was added to eachprotein so as to be a predetermined concentration. These ejectionliquids were evaluated by the same ejection experiment as in Example 1.Formulations and results examined on the present examples are listed inthe following Table 2.

TABLE 2 Compound having Ejection Protein betaine skeleton Surfactant andadditive property Type Concentration Type Concentration TypeConcentration Evaluation Example 13 insulin 4 mg/ml cocamidepropyl 3mg/ml none — ∘ betaine Example 14 insulin 4 mg/ml lauramidepropyl 3mg/ml none — ∘ betaine Comparative insulin 4 mg/ml none — TWEEN20 3mg/ml x Example 14

Although the necessary concentration of alkylamide propyl betaine to beadded varies with the concentration and type of protein, the addition ofalkylamide propyl betaine makes each protein normally ejected on thebasis of the principle of the thermal ink jet system, and it wasconfirmed that alkylamide propyl betaine exerts the effect to a widerange of protein with a small amount. As a result of an HPLC analysis,Examples 13 and 14 did not show a change of a peak chart before andafter ejection, and a change of a liquid composition.

Examples 15 to 19

(Synergistic Effect of Compound Having Betaine Skeleton and Surfactant)

A solution was prepared by adding a compound having a betaine skeletoninto protein, and an ejection liquid was prepared by further adding asurfactant to the solution. These ejection liquids were evaluated by thesame ejection experiment as in Example 1. Formulations and resultsexamined on the present examples are listed in the following Table 3.

TABLE 3 Compound having Ejection Protein betaine skeleton Surfactant andadditive property Type Concentration Type Concentration TypeConcentration Evaluation Example 15 insulin 4 mg/ml lauryl betaine 2mg/ml TWEEN20 0.5 mg/ml   ∘ Example 16 albumin 1 mg/ml lauramidepropyl 5mg/ml TWEEN80 5 mg/ml ∘ betaine Example 17 albumin 1 mg/mlcocamidepropyl 5 mg/ml TWEEN80 5 mg/ml ∘ betaine Example 18 albumin 1mg/ml lauramidepropyl 3 mg/ml TWEEN80 2 mg/ml ∘ betaine Example 19albumin 1 mg/ml cocamidepropyl 3 mg/ml TWEEN80 2 mg/ml ∘ betaine

A protein solution simultaneously containing alkylamide propyl betaineand TWEEN as an additive could be ejected, even when the concentrationof a compound having a betaine skeleton is extremely low in comparisonwith the case when the compound having a betaine skeleton was singlyadded. In addition, a protein solution could be ejected even whencontaining such a concentration of a compound having a betaine skeletonas did not make the protein solution ejected, which singly contains thesame amount of it. As a result, a total amount of an additive can begreatly reduced. As a result of an HPLC analysis, Examples 15 to 19 didnot show a change of a peak chart before and after ejection, and achange of a liquid composition.

Example 20

(Preparation of Antibody Chip with Use of an Ink Jet Printer, andSensing)

A Human IL2 monoclonal antibody, a Human IL4 monoclonal antibody and aHuman IL6 monoclonal antibody were prepared into a liquid having aconcentration of 0.1 to 500 μg/mL, respectively. Each ejection liquidwas prepared by adding lauramidepropyl betaine into the liquid so thatthe concentration of the betaine is 1% (w/w). The ejection liquid wasfilled in the head of an ink jet printer (trade name: PIXUS950i,manufactured by Canon Inc.), and was ejected onto a Poly-L-Lysin-coatedslide glass. FIG. 7 shows a model view of the present example. In thefigure, reference numeral 17 denotes a substrate, reference numeral 18denotes a masking reagent, reference numeral 19 denotes a substance(such as protein and peptide) specifically reacting with a substance tobe detected, reference numeral 20 denotes a substance to be detected,reference numeral 21 denotes the substance specifically reacting withthe substance to be detected, and reference numeral 22 denotes anindicator.

The antibody which had been ejected onto a glass was incubated at 4° C.,and the glass on which the antibody was incubated was masked with 1%BSA. The glass was carefully cleaned after having had been masked, andwas supplied as an antibody chip substrate. Subsequently, a solutionwhich contains 1 μg/mL each of recombinants IL2, IL4 and IL6 that weresubstances to be detected by the chip was prepared together with 1.0%lauramidepropyl betaine (w/w), 0.5% TWEEN20 (w/w) and 0.1% BSA (w/w).The liquid was filled in the head of an ink jet printer (trade name:PIXUS950i, manufactured by Canon Inc.), and was ejected on theabove-described substrate so as to form the same pattern. After theliquid was ejected, the substrate was covered with a cover glass, andwas subjected to the reaction at 4° C. After the reaction, the substratewas carefully cleaned and dried.

Subsequently, a substrate specifically bonded with a sample was reactedwith a substrate, and the substance was indicated. A solution containinga substance specifically bonded with the sample was prepared by blendingeach 1 μg/mL biotin-indicated antibody liquid (biotinylated Human IL2monoclonal antibody, biotinylated Human IL4 monoclonal antibody andbiotinylated Human IL6 monoclonal antibody), 1.0% lauramidepropylbetaine (w/w), 0.5% TWEEN20 (w/w) and 0.1% BSA (w/w) so that eachcomponent can be its final concentration; was filled in the head of anink jet printer (trade name: PIXUS950i, manufactured by Canon Inc.); andwas ejected onto the above-described substrate so as to form the samepattern. After the liquid was ejected, the substrate was covered with acover glass, and was subjected to the reaction at 4° C. After thereaction, the substrate was carefully cleaned and dried.

A solution for indication was prepared by blending 10 μg/ml, Cy3labeling streptavidin, 1.0% lauramidepropyl betaine (w/w), 0.5% TWEEN20(w/w) and 0.1% BSA (w/w) so that each component can be the finalconcentration; then was filled in the head of an ink jet printer (tradename: PIXUS950i, manufactured by Canon Inc.); and was ejected onto theabove-described substrate so as to form the same pattern. After theliquid was ejected, the substrate was covered with a cover glass, andwas subjected to the reaction at 4° C. After the reaction, the substratewas carefully cleaned and dried.

Subsequently, a substrate on which the reaction has been finished wasirradiated with excited light, and a quantity of fluorescent signal oflight emitted from Cy3 was measured by using a fluorescence scannerhaving a filter for a transmitted wavelength of 532 nm arranged therein.As the measured result, a fluorescent signal corresponding to the typeand concentration of a sample could be detected.

The present invention is not limited to the above embodiments andvarious changes and modifications are possible within the spirit andscope of the present invention. Therefore to apprise the public of thescope of the present invention, the following claims are made.

This application claims priority from Japanese Patent Application No.2005-098749 filed on Mar. 30, 2005, which is hereby incorporated byreference herein.

The invention claimed is:
 1. An ejection method using an ejection devicehaving: an ejection head based on the principle of a thermal ink jetsystem for ejecting a liquid; a tank for accommodating the liquid; and aflow path for guiding the liquid accommodated in the tank to theejection head, comprising: supplying the liquid accommodated in the tankfrom the tank to the ejection head through the flow path; applyingthermal energy to the supplied liquid; and ejecting the liquid from theejection head, wherein the liquid comprises: lauryl betaine; insulin ina concentration of no more than 2/25 times a concentration of the laurylbetaine; and a liquid medium mainly composed of water.
 2. An ejectionmethod using an ejection device having: an ejection head based on theprinciple of a thermal ink jet system for ejecting a liquid; a tank foraccommodating the liquid; and a flow path for guiding the liquidaccommodated in the tank to the ejection head, comprising: supplying theliquid accommodated in the tank from the tank to the ejection headthrough the flow path; applying thermal energy to the supplied liquid;and ejecting the liquid from the ejection head, wherein the liquidcomprises: lauryl betaine; insulin in a concentration of no more than 2times a concentration of the lauryl betaine; polyoxyethylene sorbitanfatty acid ester in a concentration of more than 1/4 times aconcentration of the lauryl betaine; and a liquid medium mainly composedof water.